方俊民Fang, Jim-Min臺灣大學:化學研究所卓佳慶Cho, Chia-ChinChia-ChinCho2010-06-302018-07-102010-06-302018-07-102009U0001-1708200912113600http://ntur.lib.ntu.edu.tw//handle/246246/187498在生物化學領域當中，醣類分子扮演著很重要的角色。藉由不同連結方式所形成的寡醣體往往可以調節與控制整個細胞間訊息的傳遞。為了研究醣分子的性質與特性，許多醣耦合體被開發出來模擬醣分子在生物系統當中所扮演的角色，透過這樣的方式再進一步去研究部份生物系統的機制。 在這些已經發表的論文當中，建立醣耦合體的方法大部分都需要透過一系列的化學修飾，因此也造成往後應用上的限制。本篇論文當中，我們實驗室針對醣類分子開發出直接建立醣類耦合體的方法。透過碘–氨的氧化作用，醛醣可以直接轉換成醯胺類化合物。一開始的目的在於開發出新式的水相有機反應，隨著後續的發展，為了能將此反應延伸到建立醣類與具有多功能性的胺類、胺基酸乃至於蛋白質的耦合體，整個實驗系統也隨著修改。透過溶劑的篩選，酸鹼性的調整，以及實驗步驟的優先順序，醛醣可以直接與胺基酸分子以及蛋白質進行耦合反應。此外，我們也透過碘–氨反應將酮酸轉換成醯胺類化合物。ㄧ系列的碘–氨反應當中，醛醣以及酮酸分子可以被直接且選擇性地轉換成醯胺類化合物，而醣分子上的官能基如氫氧基則無氧化反應發生，寡醣分子的醣苷鍵也沒有斷裂。進行耦合的胺類分子這ㄧ部份，雙鍵官能基團、苯環、酯基、以及氨基酸的光學中心在碘–氨反應當中相當穩定。此外透過反應步驟的調整，一鍋兩步化的程序，可以將原本無法進行耦合的胺基酸順利建立起來，而這樣的調整也間接證實反應機制傾向於透過醣類內酯化後再進行開還反應。外，我們利用甘露糖進行微陣列檢測的實驗，在初步的微陣列檢測結果顯示，修飾後的甘露糖醯胺依然與甘露糖專屬的辨識蛋白質(Con A)保有些微的結合力。這ㄧ系列的結果讓我們相信透過碘–氨反應所建立起的的醣分子耦合物有機會應用到研究寡醣分子的辨識以及醣蛋白的性質。Carbohydrates are essential materials in many biological processes. They can maintain and modulate the intrinsic properties of proteins. Glycoconjugates have become popular tools to investigate the biological processes. Many conjugation methods have been developed by anchoring carbohydrates to proteins. However, some of these methods may be time-consuming and result in low overall yield due to many preparation steps. ere we report a direct conjugation method by forming a robust amide bond between amine and aldehyde on the reducing end of aldose. By using iodine, an aldose was directly coupled with an amine through an amide bond in aqueous solution. Accordingly, aldoses undergo an oxidative amidation with a variety of aliphatic amines, bifunctional amines, α-amino esters, and peptides.nder this condition, the hydroxy groups on sugar skeleton are unaffected, the glycosidic bonds of the oligosaccharides are retained, and the amidation selectively occurs at the reducing end without interfering with other functional groups, e.g., the carboxy group in glucuronic acid. Various groups, such as double bond, phenyl, and ester are also inert in this condition. Furthermore, this reaction protocol was applied to conjugation of α-keto acids with amines by a sequence of decarboxylation, oxidation, and in situ amidation. It was promising to utilize this novel conjugation method for preparation of neoglycoproteins and neoglycolipids from unprotected and unmodified carbohydrates.Acknowledgement............................................................................................................Ibstract in Chinese.......................................................................................................IIIbstract in English.........................................................................................................Vable of Contents.........................................................................................................VIIndex of Figures...........................................................................................................XIIndex of Schemes........................................................................................................XIVndex of Tables.............................................................................................................XVbbreviations............................................................................................................XVIIhapter 1. Introduction..................................................................................................1.1 Amides............................................................................................................11.2 Direct oxidative amidation from alcohols, aldehydes, and ketoacids.......4 1.2.1 Using transition metal catalysts...........................................................4 a. Nickel peroxide....................................................................................4 b. Palladium.............................................................................................5 c. Ru..........................................................................................................7 d. Rh complex.........................................................................................10 e. Cu........................................................................................................11 1.2.2 Metal free amidation..........................................................................12 a. TBHP...................................................................................................12 b. Oxone...................................................................................................13 c. Iodine...................................................................................................14 d. Hydroxylamine and ketoacid............................................................161.3 Glycocojugation...........................................................................................19 1.3.1 Reductive amidation...........................................................................19 1.3.2 Oxime linkage.....................................................................................21 1.3.3 Thiazolidine formation.......................................................................23 1.3.4 Activated aldonate..............................................................................23 1.3.5 Hydrazide linkage...............................................................................24 1.3.6 Imidazole.............................................................................................251.4 Carbohydrate microarrays.........................................................................26 1.4.1 Site-specific and covalent immobilization........................................26 1.4.2 Site-specific but noncovalent immobilization...................................27hapter 2. Results and Discussion...............................................................................292.1 Amidation of aldoses by treatment of iodine in ammonia water............322.2 Amidation of aldoses with aqueous amines...............................................35 2.2.1 Isolation and characterization of gluconamide peracetate.............36 2.2.2 Isolation and characterization of N-ethyl gluconamide..................38 2.2.3 The amount of iodine used in the oxidative amidation reactions...39 2.2.4 Some general aldoses for oxidative amdation in ethylamine solution.................................................................................................40 2.2.5 Summary of oxidative amidation of aldoses in aqeous media........422.3 Amidation of aldoses with alkylamines.....................................................43 2.3.1 Preliminary study of solvents in iodine-promoted oxidaitve amidation.............................................................................................44 2.3.2 Steric effect of the primary, secondary and tertiary amines..........47 2.3.3 Amidation of diverse aldoses with aliphatic amines........................492.4 Amidation of aldose with functional amines.............................................53 2.4.1 Amidation of aldoses with amines bearing other functionalities...53 2.4.2 Amidation of aldoses with aromatic amines: limitation of our current method...................................................................................572.5 Iodine-promoted oxidative amidation of aldose with amine HCl salt....59 2.5.1 Effect of pH value...............................................................................59 2.5.2 Optimization of iodine-promoted amidation...................................622.6 Amidation of aldoses with α-amino acid...................................................63 2.6.1 Amidaiton of aldose with HCl salts of amino esters........................64 2.6.2 One-pot two-step protocol..................................................................65 2.6.3 Selective ligation of aldose with L-lysine methyl ester.....................69 2.6.4 One-pot two-step iodine-promoted oxidative amidations with cysteine.................................................................................................722.7 Amidation of aldoses with peptides and proteins.....................................73 2.7.1 Amidation of glucose with dipeptides...............................................73 2.7.2 Glutathione..........................................................................................74 2.7.3 Iodine-promoted oxidative amidation of glucose with GSH, and GSSG...................................................................................................78 2.7.4 Iodine-promoted oxidative amidation of D-lactose with BSA........792.8 Mechanistic study........................................................................................842.9 Decaboxylative amidation of α-keto acids.................................................86 2.9.1 Decarboxylative amidation of 2-keto-L-gulonic acid.......................87 2.9.2 Decarboxylative amidation of sialic acids........................................882.10 Preliminary assay of aldonamides on microarray.................................902.11 Conclusion..................................................................................................93hapter 3. Experimental Section.................................................................................953.1 General part.................................................................................................95 3.2 General procedure for the iodine-promoted amidation of carbohydrate molecules with primary amines.................................................................963.3 General procedure for oxidative amidation of aldoses with the methyl esters of α-amino acid and peptides...........................................................973.4 Procedure for microarray assay.................................................................973.5 Synthesis and characterization of compounds..........................................99hapter 4. References.................................................................................................170ppendices (1H & 13C NMR Spectra, published paper)..........................................180289194167 bytesapplication/pdfen-US醯胺化碘醛醣酮酸amidationiodinealdoseketoacidthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/187498/1/ntu-98-D94223005-1.pdf